The effect of genistein, widely used as a specific tyrosine kinase inhibitor, on rat brain Kv1.5 channels which were stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. Genistein inhibited Kv1.5 currents at ⁢50 mV in a concentration-dependent manner, with an IC50 of 54.7⁑8.2μM and a Hill coefficient of 1.1⁑0.2. Pretreatment of Kv1.5 with protein tyrosine kinase inhibitors (10μM lavendustin A and 100μM AG1296) and a tyrosine phosphatase inhibitor (500μM sodium orthovanadate) did not block the inhibitory effect of genistein. The inhibition of Kv1.5 by genistein showed voltage-independence over the full activation voltage range positive to 0 mV. The activation (at ⁢50 mV) kinetics was significantly delayed by genistein: time constant for an activation of 1.4⁑0.2 msec under control conditions and 10.0⁑1.5 msec in the presence of 60μM genistein. Genistein also slowed the deactivation of the tail currents, resulting in a crossover phenomenon: a time constant of 11.4⁑1.3 msec and 40.0⁑4.2 msec under control conditions and in the presence of 60μM genistein, respectively. Inhibition was reversed by the application of repetitive depolarizing pulses, especially during the early part of the activating pulse. These results suggest that genistein directly inhibits Kv1.5 channels, independent of phosphotyrosine-signaling pathway.